The present invention relates to a process for the preparation of an instant agar culture medium which can immediately be used as a culture medium merely by heating the agar culture medium packed in a container for culturing microorganisms such as bacterium for a short time. More specifically, the present invention is drawn to a process for the preparation of an instant agar culture medium, characterized by dissolving under heating 10 to 80% (by weight) of a prescribed amount of agar to be used in an agar culture medium in part of a prescribed amount of water to be used for dissolving culture medium components, cooling the obtained solution, separately dispersing or dissolving the rest of agar and other culture medium components in the rest of water, mixing the cooled solution with the separately prepared dispersion or solution, adjusting the viscosity of the obtained fluid to 40 to 4,000 mPaxc2x7s, packing the resulting solution into a container, hermetically sealing the container, and sterilizing it.
In the case of culturing microorganisms such as bacterium, the preparation of an agar culture medium has generally been performed as below. That is, an ager medium is prepared by weighing a prescribed amount of water into a container such as a large Erlenmeyer flask, adding prescribed amounts of powdery culture medium components including agar therein to disperse or dissolve the components, heating the obtained dispersion or solution to dissolve the agar, applying a cotton plug to the container, and sterilizing the medium by an autoclave or the like, and a prescribed amount of the resulting fluid is transfered into a chalet or the like, cooled and used for culture the microorganisms (edited by Tokyo University, Medical Studies Institute Alumni Association, xe2x80x9cSummary of Practice of Microbiologyxe2x80x9d, pp. 34-57, Maruzen, 1989).
However, the above process has a disadvantage that the prepared culture medium is easy to contaminate and dry since the container is not completely sealed, and therefore it has a problem for storing the once prepared culture medium for a long period of time. Hence, the culture medium must be prepared before performing the culture of microorganisms, and extra working other than original studies or examination must be performed.
As techniques for solving the above problems, the following have been disclosed: a process for preparing a culture medium for microorganisms employing a sealing container comprising dissolving culture medium components in water, adjusting the pH of the obtained solution, packing the resultant solution into a pouch, and sterilizing it under pressurizing and heating (official gazette of Japanese Laid-Open Patent Publication No. 62-11089/1987); and a culture medium obtained by packing culture medium components and a prescribed amount of water into a package, sealing the package and subjecting it to a heating sterilization treatment (official gazette of Japanese Laid-Open Patent Publication No. 8-205854/1996).
However, for these prior arts, the former has problems from the viewpoints of workability in opening thereof and security from a scald since the culture medium is sealed in a bag; and for the latter, in the case of packing powdery culture medium components and water into a package, it has a problem from the viewpoint of packing operation, and in the case of dissolving under heating powdery culture medium components, it has problems from the viewpoints of the color tone of the culture medium and the deterioration of the culture medium components when the medium is sterilized.
In the above prior arts, it has been necessary to employ a process for preparing an agar culture medium just before use or a process comprising sterilizing an agar culture medium, storing it in a container for a long period of time, and dissolving it under heating at the time of use. In the case of the latter, it must be dissolved under heating by warm water or an autoclave and the like; however, since the culture medium components are heated over and over again during the preparation of the culture medium, as described above, it has disadvantages that the color tone, the gel strength of agar and nutrient components of the medium deteriorate.
The present invention provides a process for the preparation of an instant agar culture medium which can immediately be used as a culture medium merely by heating the agar culture medium packed in a container just before use for a short time, can be stored for a long period of time and little suffers from the deterioration of culture medium components.
The present invention relates to a process for the preparation of an instant agar culture medium which can be dissolved by heating for a short time just before use, characterized by dissolving under heating 10 to 80% (by weight) of a prescribed amount of agar to be used in an agar culture medium in part of a prescribed amount of water to be used for dissolving culture medium components, cooling the obtained solution, separately dispersing or dissolving the rest of agar and other culture medium components in the rest of water, mixing the cooled solution with the separately prepared dispersion or solution, adjusting the viscosity of the obtained fluid to 40 to 4,000 mPaxc2x7s, packing the resulting solution into a container, hermetically sealing the container, and sterilizing it.
According to the present invention, remarkable effects as below can be obtained: 1) a culture medium which little suffers from the deterioration of culture medium components can be provided; 2) a culture medium which can immediately be used merely by heating just before use for a short time can be provided; and 3) a culture medium which does not suffer from the change of properties due to storage for a long period of time can be provided.
The present inventors have engaged in assiduous studies upon a culture medium which little suffers from the deterioration of culture medium components in view of the above prior arts, and as a result have found that all the problems of the above prior arts can be solved by dissolving under heating part of a prescribed amount of agar in part of a prescribed amount of water, cooling the obtained solution to a temperature till the agar is not set up, separately dispersing or dissolving the rest of agar and other culture medium components in the rest of water, mixing the cooled solution with the separately prepared dispersion or solution, adjusting the viscosity of the obtained fluid, packing the resulting solution into a container, hermetically sealing the container, and sterilizing it.
It is the objective of the present invention to provide a process for the preparation of an instant agar culture medium which can immediately be used as a culture medium merely by heating the agar culture medium packed in a container just before use for a short time, can be stored for a long period of time and little suffers from the deterioration of culture medium components.
The present invention solving the above problems is a process for the preparation of an instant agar culture medium, characterized by dissolving under heating 10 to 80% (by weight) of a prescribed amount of agar to be used in an agar culture medium in 20 to 50% (by weight) of a prescribed amount of water to be used for dissolving culture medium components, cooling the obtained solution, separately dispersing or dissolving the rest of agar and other culture medium components in the rest of water, mixing the cooled solution with the separately prepared dispersion or solution, adjusting the viscosity of the obtained fluid to 40 to 4,000 mPaxc2x7s, packing the resulting solution into a container, hermetically sealing the container, and sterilizing it, and according to a preferred embodiment thereof, the viscosity of the obtained fluid is from 250 to 1,500 mPaxc2x7s, and the rate of agar to be dissolved under heating is from 30 to 60% (by weight; hereunder, same as above unless otherwise specified) of a prescribed amount of water.
Next, the present invention will be described in detail.
In the process of the present invention, the agar culture medium is generally a known culture medium containing agar or refined agar (hereunder, both being referred to as agar) to be used for culturing microorganisms, and examples thereof include a standard agar culture medium, a glucose agar culture medium and a desoxycholate agar culture medium.
From 10 to 80%, preferably from 30 to 60%, of a prescribed amount of agar to be used in an agar culture medium is added into part, preferably from 20 to 50%, of a prescribed amount of water such as refined water to be used for dissolving culture medium components, the obtained solution is heated to completely dissolve the agar, and the resulting solution is cooled to from 40 to 60xc2x0 C. The amount of agar to be dissolved, the amount of refined water to be used for dissolution and the cooling temperature differ according to components of a culture medium to be prepared, and have something to do with packing suitability at the time of mixing with a solution and packing the resulting fluid into a container to be described later.
That is, when the amount of agar to be dissolved is small, the amount of agar to be dispersed into the rest of refined water to be used for dissolving culture medium components becomes large, thereby the agar failing to completely be dissolved by a heating sterilization process. On the other hand, when the amount of agar is large, it is necessary to retain the mixed solution at a high temperature in order to maintain the fluidity of the mixed solution till the completion of packing, which adds extra heat to the culture medium components other than agar and causes the deterioration of the culture medium.
The rest of agar and the culture medium components other than agar are added into the rest of refined water to be used for dissolving the culture medium components, the mixture is heated on demand to disperse the rest of agar and dissolve the culture medium components other than agar. The culture medium components other than agar differ according to a culture medium to be prepared, and, for example, in a standard agar culture medium, 10 g of peptone, 2.5 g of yeast extract, 1 g of glucose and 15 g of agar per liter of the culture medium are employed.
Next, the above agar-dissolved solution and the rest of agar are dispersed and mixed with the solution with the culture medium components other than agar dissolved, and the viscosity of the mixed fluid is adjusted to at least 40 mPaxc2x7s, preferably from 250 to 1,500 mPaxc2x7s. The pH of the mixed fluid can be adjusted to a prescribed pH on demand. The adjustment of the viscosity can be performed by the adjustment of the amount of agar to be dissolved and the temperature of the mixed fluid. The mixed fluid adjusted to a prescribed viscosity is extremely effective in the case of packing the culture medium into a container since it has uniform dispersion and can be packed quantitatively.
A container to be packed with the above mixed fluid may be any container so far as it is a container prepared from a material capable of standing heating by sterilization of a next process; however, a bottle-type one is particularly preferred since it is easy to handle.
An instant agar culture medium of the present invention can be obtained by packing the mixed fluid into the above container, hermetically sealing the container, sterilizing it under heating, and leaving it to cool. The packing of the culture medium into the container can be performed employing, for example, a cylinder packing machine, a rotary packing machine, and the sealing of the container can be performed according to an ordinary method, for example, a method of heat sealing employing a plastic film, an aluminum film, and a method of sealing by providing a cap with a packing. The heating sterilization of the culture medium can be performed according to an ordinary method employing an autoclave and a retort sterilizer. The agar dispersed in the mixed fluid is dissolved by the heating sterilization process of the culture medium to obtain a uniform solution.
As described above, the process of the present invention is characterized in that a culture medium is prepared by one-time heating sterilization, which differs from a conventional process comprising once dissolving under heating agar and culture medium components other than agar, packing the obtained fluid into a container, hermetically sealing the container, and further sterilizing it under heating, namely, a method performing heating twice. Hence, an agar culture medium excellent in quality can be prepared without the deterioration of culture medium components due to excess heating and the deterioration of the gel strength of agar.
A culture medium prepared according to the process of the present invention is heated for about 30 to 60 minutes according to the capacity of the packed container just before use to be liquid and can immediately be used for culturing microorganisms.